Respiratory exposure to fine particulate matter (PM2.5), including diesel exhaust particles (DEP), is associated with pulmonary inflammation and cardiovascular diseases. This study investigated the protective effects and underlying mechanisms of retinoic acid (RA) against PM2.5-induced lung injury and accelerated atherosclerosis. Differentially expressed genes (DEGs) from lung tissues of patients with chronic obstructive pulmonary disease (COPD) were analyzed using the Gene Expression Omnibus (GEO) database and L1000CDS², which identified RA as a candidate therapeutic compound. The Comparative Toxicogenomics Database (CTD) suggested the eNOS/NO signaling pathway as a potential mechanism for experimental validation. ApoE−/− mice were exposed to DEP via a nebulization chamber and treated orally with all-trans retinoic acid (ATRA; 1 or 5 mg/kg/day) for 8 weeks. RA significantly reduced CXCL1/2 levels in bronchoalveolar lavage fluid and pulmonary expression of Muc5ac, IL-1β, IL-6, and TNF-α. RA also lowered blood pressure, improved serum lipid profiles, and attenuated inflammatory cell infiltration, arterial wall thickening, and endothelial ultrastructural damage. In HUVECs, RA increased GTP cyclohydrolase-1 (GTPCH-1), p-eNOS, BH4 levels, and the BH4/BH2 ratio. These findings suggest that RA attenuates PM2.5-induced pulmonary inflammation and accelerated atherosclerosis by restoring endothelial homeostasis through modulation of the eNOS/NO coupling pathway.